Using repetitive sequences for DNA quality assessments

ABSTRACT

The present disclosure provides methods, arrays and kits for assessing the quality of genomic DNA samples, especially those obtained from formalin-fixed paraffin-embedded (FFPE) samples. The methods, arrays and kits provided herein use primer pairs specific to regions in the genomes of the organisms from which genomic DNA samples are obtained that have identical or nearly identical copies distributed across multiple chromosomes.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 830109_404_SEQUENCE_LISTING.txt. The text file is 4.6 KB, was created on Mar. 8, 2014, and is being submitted electronically via EFS-Web.

BACKGROUND

Technical Field

The present disclosure relates to methods, arrays and kits for assessing the quality of DNA samples.

Description of the Related Art

DNA has become biomarkers with the most potential. Compared to other biomarkers, DNA is stable and has less dynamic change. In addition, DNA as biomarkers is sequence based and has less ambiguity. Recently, discovery of DNA as biomarkers has been propelled by the growing application of next generation sequencing (NGS).

The quality of any scientific data is directly proportional to that of the starting samples. It is important to assess the quality of a starting DNA sample for downstream molecular analyses, such as qPCR assays and NGS. A bad quality sample will generate unusable data, which causes waste in material and labor. Not knowing the DNA quality can also lead to misuse of precious sample, which once used, cannot be recovered.

Various methods are known for DNA quality evaluation. Most commonly used spectrometrical methods can address some of the questions in DNA concentration and contaminants. However, it cannot detect many contaminants easily or evaluate the actual impact of contaminants in the molecular assays. Electrophoresis can evaluate the sizes of DNA sample. However, it cannot evaluate base damages, cross-linkage, modifications, which can adversely affect downstream enzymatic assays. Other people select to test a group of gene assays in PCR to estimate actual sample performance in molecular assays. However, selection of a few gene assays is sometimes biased, affected by biological changes in local chromosomal structure (as seen in pathological conditions) and does not have broader representations. Thus, the prediction for a performance of DNA samples in downstream molecular applications has been challenging, especially for DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples. Accordingly, there is a need to establish an objective method to evaluate the quality of DNA for downstream DNA analyses.

SUMMARY

In one aspect, the present disclosure provides a method for assessing the quality of a test genomic DNA sample, comprising:

(a) performing one or more real-time PCR reactions that use genomic DNA in a test genomic DNA sample as templates in the presence of one or more primer pairs, wherein each of the one or more primer pairs is specific for amplifying identical or nearly identical genomic DNA fragments that are present at multiple locations in the genome of the organism from which the DNA sample is obtained,

(b) performing one or more real-time PCR reactions that use genomic DNA in a control genomic DNA sample as templates in the presence of the one or more primer pairs used in step (a),

(c) determining the Ct values for the one or more real-time PCR reactions in step (a), and

(d) determining the Ct values for the one or more real-time PCR reactions in step (b),

wherein the difference between the Ct values determined in step (c) and the corresponding Ct values determined in step (d) for the one or more real-time PCR reactions are indicative of the quality of the test genomic DNA sample.

In certain embodiments, the number of the primer pairs is 4-8.

In certain embodiments, the genomic DNA fragments amplified in the presence of each primer pair are present at 10 or more different locations in the genome of the organism from which the DNA sample is obtained.

In certain embodiments, the genomic DNA fragments amplified in the presence of each primer pair in combination are present in more than 80% of all autosomes of the organism from which the DNA sample is obtained.

In certain embodiments, the test genomic DNA sample is obtained from human cells or tissue.

In certain embodiments, the test genomic DNA sample is obtained from a clinical sample.

In certain embodiments, the test genomic DNA sample is obtained from a formalin fixed and paraffin-embedded (FFPE) sample.

In certain embodiments, the genomic DNA fragments amplified in step (a) are between about 100 to 400 bp in length.

In certain embodiments, the genomic DNA fragments amplified in step (a) are of at least 2 substantially different sizes.

In certain embodiments, multiple real-time PCR reactions are performed in each of steps (a) and (b), and the average difference between the Ct values determined in step (c) and the corresponding Ct values determined in step (d) for two or more of the multiple real-time PCR reactions is used to assess the quality of the test genomic DNA sample.

In certain embodiments, the primer pairs are selected from the following primer pairs: (1) SEQ ID NOS:1 and 2, (2) SEQ ID NOS:3 and 4, (3) SEQ ID NOS:5 and 6, (4) SEQ ID NOS:7 and 8, (5) SEQ ID NOS:9 and 10, (6) SEQ ID NOS:11 and 12, (7) SEQ ID NOS:13 and 14, (8) SEQ ID NOS:15 and 16, (9) SEQ ID NOS:17 and 18, (10) SEQ ID NOS:19 and 20, and (11) SEQ ID NOS:21 and 22, (12) SEQ ID NOS:23 and 24.

In certain embodiments, the method further comprises performing additional real-time PCR and/or NGS analysis of the test genomic DNA sample.

In another aspect, the present disclosure provides an array for assessing the quality of a test genomic DNA sample, comprising a solid support and multiple compartments in the solid support, wherein a first primer pair specific to a first genomic DNA fragment in the test genomic DNA sample is contained in a first compartment or each of a first set of compartments, and wherein (a) the first genomic DNA fragment and (b) one or more fragments nearly identical to the first genomic DNA fragment, if present in the genome of the organism from which the DNA sample is obtained are located at multiple sites in the genome.

In certain embodiments, the array further comprises a second compartment or a second set of compartments, wherein a second primer pair specific to a second genomic DNA fragment in the test genomic DNA is contained in the second compartment or each of the second set of compartments, and wherein (a) the second genomic DNA fragment and (b) one or more fragments nearly identical to the second genomic DNA fragment, if present in the genome of the organism from which the DNA sample is obtained are located at multiple sites in the genome.

In certain embodiments, the array further comprises a third compartment or a third set of compartments, wherein a third primer pair specific to a third genomic DNA fragment in the test genomic DNA is contained in the third compartment or each of the third set of compartments, and wherein (a) the third genomic DNA fragment and (b) one or more fragments nearly identical to the third genomic DNA fragment, if present in the genome of the organism from which the DNA sample is obtained are located at multiple sites in the genome.

In certain embodiments, the array further comprises a fourth compartment or a fourth set of compartments, wherein a fourth primer pair specific to a fourth genomic DNA fragment in the test genomic DNA is present in the fourth compartment or each of the fourth set of compartments, and wherein (a) the fourth genomic DNA fragment and (b) one or more fragments nearly identical to the fourth genomic DNA fragment, if present in the genome of the organism from which the DNA sample is obtained are located at multiple sites in the genome.

In certain embodiments, the first, second, third, and fourth primer pairs if present in the array are selected from the following primer pairs: (1) SEQ ID NOS:1 and 2, (2) SEQ ID NOS:3 and 4, (3) SEQ ID NOS:5 and 6, (4) SEQ ID NOS:7 and 8, (5) SEQ ID NOS:9 and 10, (6) SEQ ID NOS:11 and 12, (7) SEQ ID NOS:13 and 14, (8) SEQ ID NOS:15 and 16, (9) SEQ ID NOS:17 and 18, (10) SEQ ID NOS:19 and 20, and (11) SEQ ID NOS:21 and 22, (12) SEQ ID NOS:23 and 24.

In another aspect, the present disclosure provides a kit for assessing the quality of a test genomic DNA sample, comprising: one or more primer pairs specific to one or more genomic DNA fragments in a test genomic DNA sample, wherein for each of the one or more genomic DNA fragments, (a) the genomic DNA fragment itself and (b) one or more fragments nearly identical to the genomic DNA fragment, if present in the genome of the organism from which the test genomic DNA sample is obtained, are located at multiple sites in the genome.

In certain embodiments, the number of the primer pairs is 4 to 8.

In a related aspect, the present disclosure provides a kit for assessing the quality of a test genomic DNA sample, comprising the array provided herein.

In certain embodiments, the kit further comprises a control genomic DNA sample.

In certain embodiments, the kit further comprises one or more reagents for performing real-time PCR.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a DNA quality control PCR array plate layout.

FIG. 2 shows correlation between results from DNA Quality Control (QC) Panel analysis and NGS sequencing results. FFPE samples that generated successful sequencing data are indicated by empty bars and those that did not are shown by solid bars.

FIG. 3 shows the FFPE-1 sample's amplicon bias toward smaller read. The left graph is a read length histogram of control genomic DNA, and the right graph is a read length histogram of the FFPE-1 sample.

DETAILED DESCRIPTION

The present disclosure provides methods, arrays and kits for assessing the quality of genomic DNA samples for downstream DNA analyses. The methods provided herein analyze regions in the genome from which a genomic DNA sample is obtained that have identical or nearly identical copies randomly distributed across multiple or all autosomes. By designing PCR assays for such regions, these methods assess sample DNA quality at many different chromosomal locations, and the performance of such PCR assays is thus less affected by genomic heterogeneity in the genomic DNA population in the sample. In addition, the PCR assays may optionally be designed to generate amplicons of different sizes. By comparing the performance of such PCR assays, the distribution of amplifiable fragment in the genomic DNA sample may be evaluated. This information can help design the optimal molecular assay for a sample with suboptimal quality.

In the following description, any ranges provided herein include all the values in the ranges unless otherwise indicated.

It should also be noted that the term “or” is generally employed in its sense including “and/or” (i.e., to mean either one, both, or any combination thereof of the alternatives) unless the content clearly dictates otherwise.

Also, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise.

As used herein, “about” means±15% of the indicated range or value unless otherwise indicated.

Genomic DNA Samples

The term “DNA” refers to a polymer comprising deoxyribonucleosides that are covalently bonded, typically by phosphodiester linkages between subunits. This term includes but is not limited to genomic DNA (DNA in the genome of an organism), cDNA (DNA reversely transcribed from mRNA), and plasmid DNA.

A “genomic DNA sample” is a sample that comprises genomic DNA isolated from a source of interest.

Genomic DNA samples whose quality may be assessed by the methods provided herein include genomic DNA samples prepared from any samples that comprise genomic DNA. Exemplary samples from which genomic DNA samples may be prepared include, but are not limited to, blood, swabs, body fluid, tissues including but not limited to, liver, spleen, kidney, lung, intestine, brain, heart, muscle, pancreas, cell cultures, plant tissues or samples, as well as lysates, extracts, or materials and fractions obtained from the samples described above or any cells and microorganisms and viruses that may be present on or in a sample and the like.

Materials obtained from clinical or forensic settings that contain nucleic acids are also within the intended meaning of the term “sample” from which a genomic DNA sample may be prepared. Preferably, the sample is a biological sample derived from a human, animal, plant, bacteria or fungi. The term “sample” also includes processed samples including preserved, fixed and/or stabilized samples, such as formalin fixed and paraffin-embedded (FFPE samples) and other samples that were treated with cross-linking fixatives such as glutaraldehyde.

Genomic DNA samples whose quality may be assessed by the methods provided herein may be prepared from nucleic acid-containing samples by any methods known in the art. Exemplary methods include lysis of nucleic acid-containing samples followed by isolating genomic DNA using, for example, organic solvent such as phenol or nucleic acid binding columns. Genomic DNA samples may also be treated with RNase to reduce or eliminate RNA contamination.

Downstream Analyses

The methods for assessing the quality of genomic DNA samples provided herein are useful in determining whether a particular genomic DNA sample is suitable for downstream molecular analyses, including NGS and real-time PCR assays. Thus, various methods for assessing the quality of genomic DNA samples provided herein may further comprise performing one or more additional analyses of the genomic DNA samples whose quality has been assessed as suitable for such analysis. As described in detail below, assessing the quality of a genomic DNA sample includes determining amplification efficiency and size distribution of amplifiable fragments of the sample.

Methods for Assessing Genomic DNA Quality

The present disclosure provides a method for assessing the quality of a test genomic DNA sample, comprising: (a) performing one or more real-time PCR reactions that use genomic DNA in a test genomic DNA sample as templates in the presence of one or more primer pairs, wherein each of the one or more primer pairs is specific for amplifying identical or nearly identical genomic DNA fragments that are present at multiple locations in the genome of the organism from which the DNA sample is obtained, (b) performing one or more real-time PCR reactions that use genomic DNA in a control genomic DNA sample as templates in the presence of the one or more primer pairs used in step (a), (c) determining the Ct values for the one or more real-time PCR reactions in step (a), and (d) determining the Ct values for the one or more real-time PCR reactions in step (b), wherein the difference between the Ct values determined in step (c) and the corresponding Ct values determined in step (d) for the one or more real-time PCR reactions are indicative of the quality of the test genomic DNA sample.

The quality of a test genomic DNA sample refers to characteristics of the test genomic DNA sample related to downstream analyses of the sample, such as genomic DNA concentration, presence of contaminants, genomic DNA sizes, degree of degradation, presence of base damages, cross-linkage, or modifications, amplification efficiency, size distribution of amplifiable fragments, and the like.

A method for assessing the quality of a test genomic DNA sample provided herein may assess one or more characteristics of the test genomic DNA sample. For example, the method may be able to characterize the amplification efficiency of a test genomic DNA sample, and thus suggest appropriate amounts of the samples to be used in downstream analyses. The method may also be able to characterize the size distribution of amplifiable fragments, which may guide the design of downstream analyses to achieve optimal use of the sample.

The method provided herein uses one or more primer pairs each of which is specific for amplifying identical or nearly identical genomic DNA fragments that are present at multiple locations in the genome of the organism from which the genomic DNA sample is obtained. The presence of the identical or nearly identical genomic DNA fragments at multiple locations in the genome provides a broader representation of the genomic DNA population in the genomic DNA sample than a genomic DNA fragment present only at one location in the genome. Thus, the overall amplification performance using such primer pair(s) is less affected by genomic heterogeneity in the genomic DNA population of the sample.

For example, in the Example described below, the primer pair for Primer Assay No. 1 (SEQ ID NOS:1 and 2) is able to amplify a 111 bp genomic DNA fragment that is present in 20 locations in the human genome and another 110 bp nearly identical sequence at another location in the human genome. Thus, using this primer pair, genomic DNA amplification at 21 different locations in the human genome may be analyzed.

A genomic DNA fragment is nearly identical to another DNA if (a) the size difference between the two genomic DNA fragments is at most 5% (e.g., at most 4%, 3%, 2% or 1%) of the full length of the longer fragment, and (b) the sequence identity between the two fragments is at least 95% (e.g., at least 96%, 97%, 98%, or 99%).

Preferably, genomic DNA fragments amplified in the presence of a primer pair only have at most 4% differences in size and in sequence between each other. More preferably, genomic DNA fragments amplified in the presence of a primer pair only have at most 2% differences in size and in sequence between each other.

While any method known in the art for making such determinations may be used, for the purpose of the present invention, the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990) and Karlin et al., PNAS USA 90:5873-5787 (1993) is used for determining sequence identity according to the methods of the invention. A particularly useful BLAST program is the WU-BLAST-2 program (Altschul et al., Methods in Enzymology 266:460-480 (1996)). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. A percent nucleic acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the “longer” sequence in the aligned region. The “longer” sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).

An “oligonucleotide” refers to a short polymer composed of deoxyribonucleotides, ribonucleotides or combinations thereof. Oligonucleotides are generally between about 10 to 100 nucleotides, preferably about 15 to 30 nucleotides, in length.

A “primer” for amplification is an oligonucleotide that is complementary to a target nucleotide sequence and leads to addition of nucleotides to the 3′ end of the primer in the presence of a DNA or RNA polymerase.

A primer pair “specific for amplifying” a target sequence or a primer pair “specific to” a target sequence refers to a primer pair capable of specifically amplifying the target sequence.

“Specifically amplifying” a target sequence means amplifying the target sequence or a sequence that is nearly identical to the target sequence without amplifying other sequences in a reaction mixture.

A sequence that is “nearly identical” to a target sequence if the size difference between these two sequences are at most 5% of the longer fragment and the sequence identity between these two sequences is at least 95%.

The genomic DNA fragments amplified in the presence of a particular primer pair may be present at 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more different locations in a genome of interest. They may be present at 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different autosomes. Preferably, no more than 50%, 40%, 30% or 20% of the genomic DNA fragment amplified by the particular primer pair are located on a single chromosome.

The genomic DNA fragments amplified by multiple primer pairs may be present in more than 50%, 60%, 70%, 80%, 85%, 90%, or 95% of all autosomes of the organism from which a genomic DNA sample is obtained. Preferably, the genomic DNA fragments amplified are not present on sex chromosomes. Also preferably, no more than 50%, 40%, 30% or 20% of the genomic DNA fragment amplified by the multiple primer pairs are located on a single chromosome.

Primer pairs used in the method provided herein may be designed by identifying regions in a genome of interest regions that have identical or highly nearly identical copies distributed on different chromosomes using bioinformatic approach. Preferably, such regions do not contain known single nucleotide polymorphisms (SNPs), insertions or deletions (INDELs), repetitive sequences, or other variations.

The method provided herein may use 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more primer pairs.

In one embodiment, two primer pairs may share a primer so that the shorter amplicons produced by one primer pair are completely within the longer amplicons produced by the other primer pair. This configuration allows evaluation of the frequency of random DNA damages occurred in the sample.

The genomic DNA fragments amplified by real-time PCR may be from about 60 to 600 bp, such as about 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 450, 500, or 550 bp.

The sizes of the genomic DNA fragments amplified by different primer pairs may be the same or different. For example, a method provided herein may use primer pairs that generate amplicons that are about 100, 200, 300 and 400 bp. It is also contemplated that multiple primer pairs are used to produce amplicons of the same or a similar size. For example, the method in the Example used 12 primer pairs: a first set of 3 primer pairs amplified genomic DNA fragments of about 100 bp, a second set of 3 primer pairs amplified genomic DNA fragments of about 200 bp, a third set of 3 primer pairs amplified genomic DNA fragments of about 300 bp, and a fourth set of 3 primer pairs amplified genomic DNA fragments of about 400 bp. The use of primer pairs that amplify genomic DNA fragments of different sizes allows the evaluation of the distribution of amplifiable fragments in a genomic DNA sample, which in turn guides the best use of samples of inferior quality.

To ensure broad applicability of the method provided herein, the primer pairs may be tested to assess population variability. For example, if the method is for assessing the quality of human genomic DNA samples, candidate primer pairs may be used to analyze genomic DNA samples from different human populations to evaluate their consistency. If the method is for assessing the quality of mouse genomic DNA samples, candidate primer pairs may be used to analyze genomic DNA samples from different mouse strains.

In one embodiment, the method provided herein is to assess the quality of genomic DNA samples prepared from samples that contain human cells or tissues. The method may use one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) of the following primer pairs: (1) SEQ ID NOS:1 and 2, (2) SEQ ID NOS:3 and 4, (3) SEQ ID NOS:5 and 6, (4) SEQ ID NOS:7 and 8, (5) SEQ ID NOS:9 and 10, (6) SEQ ID NOS:11 and 12, (7) SEQ ID NOS:13 and 14, (8) SEQ ID NOS:15 and 16, (9) SEQ ID NOS:17 and 18, (10) SEQ ID NOS:19 and 20, (11) SEQ ID NOS:21 and 22, and (12) SEQ ID NOS:23 and 24). The sequences of such primer pairs are provided in the Example below.

When multiple primer pairs are used, each primer pair may be included in an individual real-time PCR reaction. Alternatively, multiple primer pairs may be included in a single real-time PCR reaction. For example, some primer pairs may be included in a single PCR reaction, and the other primer pairs may be included in one or more other PCR reactions. In one embodiment, two or more PCR primers that amplify genomic DNA fragments of the same or a similar size are included in a single PCR reaction. A genomic DNA fragment is of a similar size as another genomic DNA fragment if the difference between the two fragments is less than 5% (e.g., less than 4%, 3%, 2% or 1%) of the longer fragment.

Preferably, the test genomic DNA sample is from a sample that comprises human cells or tissue, such as a clinical sample. Exemplary preferred primer pairs for assessing the quality of human genomic DNA samples are provided in the Example.

A control genomic DNA sample is a genomic DNA sample isolated from a sample that contains cells or tissue from the same species as the sample from which a test genomic DNA sample is isolated. In addition, the control genomic DNA sample has been shown to be of high quality by, for example, real-time PCR, NGS or other analyses.

For example, if a method is to assess the quality of certain genomic DNA samples prepared from human FFPF tissues, a control genomic DNA sample may be a human genomic DNA sample prepared from human blood and has been shown to be of good quality (e.g., with high amplification efficiency and producing high quality NGS data).

“Real-time polymerase chain reaction (PCR)” (also referred to as “qPCR”) refers to a type of PCR that amplifies and simultaneously quantify a target DNA molecule. Its key feature is that the amplified DNA is detected as the reaction progresses in real time.

Similar to traditional PCR reactions, real-time PCR reaction mixtures contain DNA polymerase, such as Taq DNA polymerase (e.g., hot-start Taq DNA polymerase), buffer, magnesium, dNTPs, and optionally other agents (e.g., stabilizing agents such as gelatin and bovine serum albumin). In addition, real-time PCR reaction mixtures also contain reagents for real time detection and quantification of amplification products.

For real-time detection and quantification, probes specific to amplification products may be detectably labeled with a fluorophore. Alternatively, the amplification reaction may be performed in the presence of an intercalating dye. Changes in fluorescence during the amplification reaction are monitored and are used in measuring the amount of amplification products.

Exemplary fluorophore-labeled probes include TAQMAN® probes. Such a probe is typically labeled with a reporter molecule such as a fluorescent dye at its 5′ end and a quencher molecule at its 3′ end. The close proximity of the reporter molecule to the quencher molecule prevents detection of its fluorescence. Breakdown of the probe by the 5′ to 3′ exonuclease activity of a DNA polymerase (e.g., Taq polymerase) breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected. An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter. Additional exemplary fluorophore-labeled probes similar to TAQMAN® probes include Molecular Beacon probes and Scorpion probes.

Exemplary intercalating dyes include SYBR® Green. Such a dye binds to all double-stranded DNA in PCR, causing fluorescence of the dye. An increase in DNA product during PCR thus leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentration to be quantified.

DNA quantification by real-time PCR may rely on plotting fluorescence against the number of cycles on a logarithmic scale. A threshold for detecting DNA-based fluorescence is set slightly above background. The number of cycles at which the fluorescence exceeds the threshold is called “the threshold cycle (Ct).”

Preferably, one or more real-time PCR reactions that use genomic DNA in a test genomic DNA sample as templates performed in step (a) of the method provided herein are performed under the same or similar conditions as the real-time PCR reactions that use genomic DNA in a control genomic DNA sample as templates are performed in step (b). For example, the real-time PCR reactions of steps (a) and (b) may be performed in reaction mixtures containing the same DNA polymerase in the same amount, the same PCR buffer, the same magnesium concentration, the same intercalating dye in the same amount if the intercalating dye is used for detection and quantification of amplification products, and the same dNTPs concentration and under the same thermocycling scheme.

Also preferably, the Ct values for the one or more real-time PCR reactions in step (a) determined in step (c) of the method provided herein are determined under the same or similar conditions as the Ct values for the one or more real-time PCR reactions in step (b) are determined in step (d). For example, the Ct determination in steps (c) and (d) may be performed using the same apparatus.

A Ct value determined in step (d) “corresponds to” a Ct value determined in step (c) if the Ct value determined in step (d) is obtained from a reaction mixture that contains the same primer pair or the same set of primer pairs as the reaction mixture from which the Ct value determined in step (c) is obtained. In other words, the Ct value determined in step (c) and the “corresponding” Ct value determined in step (d) are to quantify genomic DNA fragments amplified in the presence of the same primer pair or the same set of primer pairs.

The difference between a Ct value determined in step (c) and the corresponding Ct value determined in step (d) may be used to characterize or indicate the quality of the test genomic DNA sample. For example, the difference equal to or less than a predetermined value may indicate that the test genomic DNA sample is of high quality that is suitable for downstream analysis (e.g., qPCR and NGS), whereas the difference more than a predetermined value may indicate that the test genomic DNA sample is of low quality. The predetermined value for a given primer pair may be obtained using control samples and other samples for which downstream analyses have been performed.

If a method for assessing the quality of a test genomic DNA sample comprises amplifying genomic DNA fragments using multiple primer pairs, the Ct difference between real-time PCR reactions using genomic DNA in a test genomic DNA sample as templates and those using genomic DNA in a control genomic DNA sample as templates for each primer pair may be determined and used to characterize or indicate the quality of the test genomic DNA sample. In addition, the average of the Ct differences for the multiple primer pairs may be used to characterize or indicate the overall quality of the test genomic DNA sample.

The method for assessing the quality of a test genomic DNA sample may comprise amplifying genomic DNA fragments using multiple primer pairs that produce amplicons of the same or a substantially similar size but with substantially different sequences. Amplicons are similar in size if the size differences among these amplicons are less than about 25% of the longest amplicon. Amplicons are substantially different in sequences if sequence identities among these amplicons are less than about 50%. In such a case, the Ct difference between real-time PCR reactions using genomic DNA in a test genomic DNA sample as templates and those using genomic DNA in a control genomic DNA sample as templates for each primer pair may be determined and used to characterize or indicate the quality of the test genomic DNA sample. In addition, the average of the Ct differences for the multiple primer pairs may be used to characterize or indicate the quality of the test genomic DNA sample with respect to amplifying genomic DNA fragments that are similar in size as those amplified using the primer pairs. If the method also uses other primer pairs that amplify genomic DNA fragments that are substantially different in size from those described above, the average of the Ct differences for all the primer pairs used may be determined and used to characterize or indicate the overall quality of the test genomic DNA sample.

The method for assessing the quality of a genomic DNA sample may use multiple primer pairs that amplify genomic DNA fragments having at least 2, 3, 4, or 5 substantially different sizes. Two genomic DNA fragments are substantially different in size if their size different is more than 50 bp, such as more than 75 bp, 100 bp, 125 bp, or 150 bp. For example, in the method described below in the Example, 12 primer pairs were used to produce genomic DNA fragments having 4 substantially different sizes: about 100 bp, about 200 bp, about 300 bp and about 400 bp.

Arrays for Assessing Genomic DNA Quality

The present disclosure also provides an array for assessing the quality of a DNA sample.

The array comprises a solid support and multiple compartments in the solid support. Exemplary arrays include multi-well plates, such as 96-well, 100-well, 384-well plates, and the like. The layout of an exemplary array is shown in FIG. 1.

The array provided herein may comprise a first primer pair specific to a first genomic DNA fragment in a test genomic DNA sample in a first compartment or each of a first set of compartments, and wherein (a) the first genomic DNA fragment and (b) one or more fragments nearly identical to the first genomic DNA fragment, if present in the genome of the organism from which the DNA sample is obtained are located at multiple sites in the genome.

The array may further comprise a second compartment or a second set of compartments, wherein a second primer pair specific to a second genomic DNA fragment in the test genomic DNA is contained in the second compartment or each of the second set of compartments, and wherein (a) the second genomic DNA fragment and (b) one or more fragments nearly identical to the second genomic DNA fragment, if present in the genome of the organism from which the DNA sample is obtained are located at multiple sites in the genome.

The array may further comprise a third compartment or a third set of compartments, wherein a third primer pair specific to a third genomic DNA fragment in the test genomic DNA is contained in the third compartment or each of the third set of compartments, and wherein (a) the third genomic DNA fragment and (b) one or more fragments nearly identical to the third genomic DNA fragment, if present in the genome of the organism from which the DNA sample is obtained are located at multiple sites in the genome.

The array may further comprise a fourth compartment or a fourth set of compartments, wherein a fourth primer pair specific to a fourth genomic DNA fragment in the test genomic DNA is present in the fourth compartment or each of the fourth set of compartments, and wherein (a) the fourth genomic DNA fragment and (b) one or more fragments nearly identical to the fourth genomic DNA fragment, if present in the genome of the organism from which the DNA sample is obtained are located at multiple sites in the genome.

The array may further comprise one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 20, and 24) additional compartments or one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 20, and 24) additional set of compartments that contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 20, and 24) primer pairs. Each of the additional primer pairs is able to amplify identical or nearly identical genomic DNA fragments that are present at multiple sites in the genome of the organism from which the test genomic DNA sample is obtained.

If a set of compartments are present in an array that each contain the same primer pair, one of the compartments may contain, in addition to the primer pair, a portion of a control genomic DNA sample.

The primer pairs that may be included in the array provided herein and control genomic DNA samples are described above with respect to methods for assessing the quality of a test genomic DNA sample.

In a related aspect, the present disclosure also provides use of the array provided herein in assessing the quality of a genomic DNA sample, including determining suitability of using the genomic DNA sample in downstream analysis such as NGS and additional real-time PCR analysis and determining the size distribution of amplifiable fragments in the genomic DNA sample.

Kits for Assessing Genomic DNA Quality

The present disclosure also provides kits for assessing the quality of a genomic DNA sample.

The kit provided herein comprises one or more primer pairs specific to one or more genomic DNA fragments in a test genomic DNA sample, wherein for each of the one or more genomic DNA fragments, (a) the genomic DNA fragment itself and (b) one or more fragments nearly identical to the genomic DNA fragment, if present in the genome of the organism from which the test genomic DNA sample is obtained, are located at multiple sites in the genome. The number of the primer pairs in a kit may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more.

The one or more primer pairs may be in an array format. Thus, the kit provided herein may comprise an array for assessing genomic DNA quality as described above.

The kit may further comprise a control genomic DNA sample.

The kit may further comprise one or more reagents for performing real-time PCR.

The reagents include buffer, magnesium, dNTPs, DNA polymerase (e.g., hot start polymerase), and other agents (e.g., stabilizing agents such as gelatin and bovine serum albumin). Some of the reagents (e.g., dNTPs, magnesium, and buffer) may be pre-mixed to form a PCR reaction mix that is included in the kit.

The primer pairs that may be included in the kit provided herein and control genomic DNA samples are described above with respect to methods for assessing the quality of a test genomic DNA sample.

In a related aspect, the present disclosure also provides use of the kit provided herein in assessing the quality of a genomic DNA sample, including determining suitability of using the genomic DNA sample in downstream analysis such as NGS and additional real-time PCR analysis and determining the size distribution of amplifiable fragments in the genomic DNA sample.

The following example is for illustration and is not limiting.

EXAMPLE

Using bioinformatic approach, more than a few hundred regions in the human genome that have identical or highly similar copies randomly distributed on different chromosomes were identified. Real-time PCR amplicons of approximately 100, 200, 300 and 400 bp were designed for most of them. Some shorter PCR amplicons were completely within other longer PCR amplicons, and two of the pairs share one common primer site. This configuration made it feasible to evaluate the frequency of random DNA damages occurred in the sample.

The performance of those PCR assays was verified in over a hundred normal human DNA samples representing different ethnic origins. 48 assays were selected based on the assay sensitivity and robustness in the tested population. Based on qPCR assay performance, 12 assays were selected for further evaluation.

For each of the 12 assays, the primer pair sequences, the number of sequences that the primer pair is able to amplify (referred to as “number of hits”) in the human genome, hg19 (GRCh37 Genome Reference Consortium Human Reference 37 (GCA_000001405.1)), the size of the first hit, and the location of first hit are provided in Table 1 below:

TABLE 1 SEQ Assay Primer Sequence ID No. of First Hit No. (5′→3′) NO: Hits Size First Hit Location 1 TGGTAGCTTGAGTCACTGTG 1 21 111 bp chr11:135345 + 135455 GGATTTGGGCATAGGTTTG 2 2 ATGATGGATCTTTCCCAAC 3 24 103 bp chr15:20140335 + 20140437 TGACAAGTAAAGCTGGAATAATC 4 3 TAAATCATCCACATACTGAAGGAC 5 25  92 bp chr10:66540447 + 66540538 ATAGCCCTCATCTGTTTGGTC 6 4 TTCCCACACCAGTCTTCAC 7 22 205 bp chr11:135251 + 135455 GGATTTGGGCATAGGTTTG 8 5 CCTCCCAAGTGTTCTGCTC 9 27 212 bp chr15:20140226 + 20140437 TGACAAGTAAAGCTGGAATAATC 10 6 CCTTATTATCACCCTGCTCTC 11  31* 219 bp chr10:99460360 + 99460578 CCTGTGGGTATTTCTAGTCG 12 7 CCTCACTCCCTCACTCGAC 13 27 309 bp chr11:135147 + 135455 GGATTTGGGCATAGGTTTG 14 8 TCACTCCCTCACTCGACAC 15 27 307 bp chr11:135149 + 135455 GGATTTGGGCATAGGTTTG 16 9 TATAAAGGCACTAATCCCATTC 17 22 295 bp chr15:20140175 + 20140469 TTACATAGGACAGATGCAAATAGAC 18 10 TCATCTGAGAAGGTGGAGC 19 20 380 bp chr11:135076 + 135455 GGATTTGGGCATAGGTTTG 20 11 CAAATTCAGTGTTGATGAGAGC 21 26 399 bp chr15:20140071 + 20140469 TTACATAGGACAGATGCAAATAGAC 22 12 GCCTCGTGGGATGAGAAAG 23  57* 401 bp chr10:99459976 + 99460376 GCAGGGTGATAATAAGGAGAAG 24 *The numbers of hits for Assay Nos. 6 and 12 do not include 2 hits in unplaced contigs for Assay No. 6 or 3 hits in unplaced contigs for Assay No. 12. For each assay, the oligonucleotide in the top row in the above table is the forward 5′-primer, whereas the oligonucleotide in the bottom row is the reverse 3′-primer.

The locations and sizes of the hits (that have perfect matches with 19 nucleotides at the 3′ end of primers) for each of the twelve primer pairs are provided in Tables 2-13 below.

TABLE 2 Targets for Primer Assay No. 1 Hit No. Hit Location (hg19) Hit Size (bp) 1 chr11: 135345 + 135455 111 2 chr11: 131922 + 132032 111 3 chr19: 205625 + 205735 111 4 chr19: 202211 + 202321 111 5 chr1: 243221121 + 243221231 111 6 chr1: 675964 + F676074 111 7 chr1: 672548 + 672658 111 8 chr1: 669142 + 669251 110 9 chr1: 665733 + 665843 111 10 chr1: 140245 + 140355 111 11 chr7: 56901226 + 56901336 111 12 chr10: 38736084 − 38736194 111 13 chr16: 90231916 − 90232026 111 14 chr16: 90228497 − 90228607 111 15 chr1: 323780 − 323890 111 16 chr1: 320373 − 320483 111 17 chr3: 197944687 − 197944797 111 18 chr3: 197941273 − 197941383 111 19 chr5: 180750395 − 180750505 111 20 chr5: 180746988 − 180747098 111 21 chr7: 128290584 − 128290694 111

TABLE 3 Targets for Primer Assay No. 2 Hit No. Hit Location (hg19) Hit Size (bp) 1 chr15: 20140335 + 20140437 103 2 chr16: 34178086 + 34178188 103 3 chr16: 33844143 + 33844245 103 4 chr16: 33061841 + 33061943 103 5 chr16: 32118544 + 32118646 103 6 chr2: 92265099 + 92265201 103 7 chr2: 91988683 + 91988785 103 8 chr2: 91661079 + 91661180 102 9 chr2: 90439250 + 90439351 102 10 chr7: 64575049 + 64575151 103 11 chr7: 61675244 + 61675346 103 12 chr9: 42734973 − 42735075 103 13 chr10: 42604408 − 42604510 103 14 chr16: 32831850 − 32831952 103 15 chr21: 10893340 − 10893442 103 16 chr2: 132804665 − 132804767 103 17 chr2: 132765379 − 132765481 103 18 chr2: 92240041 − 92240143 103 19 chr7: 65046135 − 65046237 103 20 chr7: 64983245 − 64983347 103 21 chr7: 57937370 − 57937472 103 22 chr9: 70411890 − 70411992 103 23 chr9: 70160962 − 70161064 103 24 chr9: 69795962 − 69796064 103

TABLE 4 Targets for Primer Assay No. 3 Hit No. Hit Location (hg19) Hit Size (bp) 1 chr10: 66540447 + 66540538 92 2 chr11: 101107279 + 101107370 92 3 chr12: 95847668 + 95847759 92 4 chr1: 94840246 + 94840337 92 5 chr2: 218982552 + 218982643 92 6 chr3: 44588305 + 44588396 92 7 chr4: 35862674 + 35862765 92 8 chr6: 87386987 + 87387078 92 9 chr7: 151729234 + 151729325 92 10 chr7: 23897603 + 23897694 92 11 chr8: 87942626 + 87942717 92 12 chr8: 54052020 + 54052111 92 13 chr8: 42419179 + 42419270 92 14 chr10: 121647046 − 121647137 92 15 chr12: 131823434 − 131823525 92 16 chr19: 8448392 − 8448483 92 17 chr1: 161224222 − 161224313 92 18 chr1: 105655701 − 105655792 92 19 chr2: 98441604 − 98441695 92 20 chr3: 190907594 − 190907685 92 21 chr3: 164533317 − 164533408 92 22 chr4: 45537496 − 45537587 92 23 chr5: 114330130 − 114330221 92 24 chr6: 38588227 − 38588318 92 25 chr6: 16459961 − 16460052 92

TABLE 5 Targets for Primer Assay No. 4 Hit No. Hit Location (hg19) Hit Size (bp) 1 chr11: 135251 + 135455 205 2 chr11: 131828 + 132032 205 3 chr19: 205531 + 205735 205 4 chr19: 202117 + 202321 205 5 chr1: 243221027 + 243221231 205 6 chr1: 675870 + 676074 205 7 chr1: 672454 + 672658 205 8 chr1: 669048 + 669251 204 9 chr1: 665639 + 665843 205 10 chr1: 140151 + 140355 205 11 chr7: 56446682 + 56446886 205 12 chr7: 55816209 + 55816413 205 13 chr16: 90231916 − 90232120 205 14 chr16: 90228497 − 90228701 205 15 chr1: 323780 − 323984 205 16 chr1: 320373 − 320577 205 17 chr3: 197944687 − 197944891 205 18 chr3: 197941273 − 197941477 205 19 chr5: 180750395 − 180750599 205 20 chr5: 180746988 − 180747192 205 21 chr7: 128290584 − 128290788 205 22 chr7: 45846431 − 45846635 205

TABLE 6 Targets for Primer Assay No. 5 Hit No. Hit Location (hg19) Hit Size (bp) 1 chr15: 20140226 + 20140437 212 2 chr16: 34177977 + 34178188 212 3 chr16: 33844034 + 33844245 212 4 chr16: 33061732 + 33061943 212 5 chr16: 32118435 + 32118646 212 6 chr2: 92264990 + 92265201 212 7 chr2: 91988574 + 91988785 212 8 chr2: 91660970 + 91661180 211 9 chr2: 90439141 + 90439351 211 10 chr7: 64574941 + 64575151 211 11 chr9: 42734864 + 42735075 212 12 chr10: 42615575 − 42615786 212 13 chr10: 42604408 − 42604618 211 14 chr16: 46469617 − 46469827 211 15 chr16: 46458916 − 46459127 212 16 chr16: 32831850 − 32832061 212 17 chr21: 10893340 − 10893551 212 18 chr2: 132804665 − 132804876 212 19 chr2: 132765379 − 132765590 212 20 chr2: 92240041 − 92240252 212 21 chr7: 65046135 − 65046345 211 22 chr7: 64983245 − 64983455 211 23 chr7: 61761699 − 61761910 212 24 chr7: 57937370 − 57937581 212 25 chr9: 70411890 − 70412101 212 26 chr9: 70160962 − 70161173 212 27 chr9: 69795962 − 69796173 212

TABLE 7 Targets for Primer Assay No. 6 Hit No. Hit Location (hg19) Hit Size (bp) 1 chr10: 99460360 + 99460578 219 2 chr10: 29716426 + 29716644 219 3 chr12: 49510213 + 49510431 219 4 chr13: 99417362 + 99417580 219 5 chrUn_gl000223: 15304 + 15518 215 6 chrUn_gl000223: 10365 + 10582 218 7 chr1: 47599632 + 47599850 219 8 chr2: 95857874 + 95858092 219 9 chr4: 9668931 + 9669149 219 10 chr4: 9132851 + 9133068 218 11 chr4: 9124292 + F9124510 219 12 chr5: 56843254 + 56843466 213 13 chr6: 52748818 + 52749037 220 14 chr8: 8063979 + 8064196 218 15 chr9: 28880975 + F28881187 213 16 chrX: 153750154 + F153750366 213 17 chrX: 126488690 + F126488902 213 18 chr11: 67597481 − 67597698 218 19 chr11: 3468681 − 3468898 218 20 chr12: 133672083 − 133672296 214 21 chr12: 133667147 − 133667361 215 22 chr13: 114947411 − 114947629 219 23 chr15: 90889818 − 90890036 219 24 chr2: 169418786 − 169418998 213 25 chr3: 195454995 − 195455213 219 26 chr4: 157307531 − 157307749 219 27 chr4: 3987662 − 3987874 213 28 chr4: 3979076 − 3979294 219 29 chr5: 39828181 − 39828395 215 30 chr7: 67568765 − 67568983 219 31 chr7: 38270470 − 38270688 219 32 chr8: 12316517 − 12316734 218 33 chr8: 12073995 − 12074212 218

TABLE 8 Targets for Primer Assay No. 7 Hit No. Hit Location (hg19) Hit Size (bp) 1 chr11: 135147 + 135455 309 2 chr11: 131724 + 132032 309 3 chr19: 205427 + 205735 309 4 chr19: 202013 + 202321 309 5 chr1: 243220923 + 243221231 309 6 chr1: 222650846 + 222651154 309 7 chr1: 675766 + 676074 309 8 chr1: 672350 + 672658 309 9 chr1: 668944 + 669251 308 10 chr1: 665535 + 665843 309 11 chr1: 140047 + 140355 309 12 chr4: 120331644 + 120331952 309 13 chr7: 56446578 + 56446886 309 14 chr7: 55816105 + 55816413 309 15 chr10: 38736084 − 38736392 309 16 chr16: 90231916 − 90232224 309 17 chr16: 90228497 − 90228805 309 18 chr1: 323780 − 324088 309 19 chr1: 320373 − 320681 309 20 chr3: 197944687 − 197944995 309 21 chr3: 197941273 − 197941581 309 22 chr4: 119550936 − 119551244 309 23 chr5: 180750395 − 180750703 309 24 chr5: 180746988 − 180747296 309 25 chr7: 128290584 − 128290892 309 26 chr7: 45846431 − 45846739 309 27 chr7: 39830686 − 39830993 308

TABLE 9 Targets for Primer Assay No. 8 Hit No. Hit Location (hg19) Hit Size (bp) 1 chr11: 135149 + 135455 307 2 chr11: 131726 + 132032 307 3 chr19: 205429 + 205735 307 4 chr19: 202015 + 202321 307 5 chr1: 243220925 + 243221231 307 6 chr1: 222650848 + 222651154 307 7 chr1: 675768 + 676074 307 8 chr1: 672352 + 672658 307 9 chr1: 668946 + 669251 306 10 chr1: 665537 + 665843 307 11 chr1: 140049 + 140355 307 12 chr4: 120331646 + 120331952 307 13 chr7: 56446580 + 56446886 307 14 chr7: 55816107 + 55816413 307 15 chr10: 38736084 − 38736390 307 16 chr16: 90231916 − 90232222 307 17 chr16: 90228497 − 90228803 307 18 chr1: 323780 − 324086 307 19 chr1: 320373 − 320679 307 20 chr3: 197944687 − 197944993 307 21 chr3: 197941273 − 197941579 307 22 chr4: 119550936 − 119551242 307 23 chr5: 180750395 − 180750701 307 24 chr5: 180746988 − 180747294 307 25 chr7: 128290584 − 128290890 307 26 chr7: 45846431 − 45846737 307 27 chr7: 39830686 − 39830991 306

TABLE 10 Targets for Primer Assay No. 9 Hit No. Hit Location (hg19) Hit Size (bp) 1 chr15: 20140175 + 20140469 295 2 chr16: 33061681 + 33061975 295 3 chr16: 32118384 + 32118678 295 4 chr2: 91660919 + 91661212 294 5 chr2: 90439090 + 90439383 294 6 chr7: 64574890 + 64575183 294 7 chr7: 61675084 + 61675378 295 8 chr9: 42734813 + 42735107 295 9 chr16: 46469585 − 46469878 294 10 chr18: 15206512 − 15206806 295 11 chr18: 15162997 − 15163291 295 12 chr21: 10893308 − 10893602 295 13 chr2: 132804633 − 132804927 295 14 chr2: 132765347 − 132765641 295 15 chr7: 65046103 − 65046396 294 16 chr7: 64983213 − 64983506 294 17 chr7: 61761667 − 61761961 295 18 chr7: 61058228 − 61058522 295 19 chr7: 57937338 − 57937632 295 20 chr9: 70411858 − 70412152 295 21 chr9: 70160930 − 70161224 295 22 chr9: 69795930 − 69796224 295

TABLE 11 Targets for Primer Assay No. 10 Hit No. Hit Location (hg19) Hit Size (bp) 1 chr11: 135076 + 135455 380 2 chr11: 131653 + 132032 380 3 chr19: 205356 + 205735 380 4 chr19: 201942 + 202321 380 5 chr1: 675695 + 676074 380 6 chr1: 672279 + 672658 380 7 chr1: 668873 + 669251 379 8 chr1: 665464 + 665843 380 9 chr1: 139976 + 140355 380 10 chr7: 56446507 + 56446886 380 11 chr7: 55816034 + 55816413 380 12 chr16: 90231916 − 90232295 380 13 chr16: 90228497 − 90228876 380 14 chr1: 323780 − 324159 380 15 chr1: 320373 − 320752 380 16 chr3: 197944687 − 197945066 380 17 chr3: 197941273 − 197941652 380 18 chr5: 180750395 − 180750774 380 19 chr5: 180746988 − 180747367 380 20 chr7: 45846431 − 45846810 380

TABLE 12 Targets for Primer Assay No. 11 Hit No. Hit Location (hg19) Hit Size (bp) 1 chr15: 20140071 + 20140469 399 2 chr16: 33061577 + 33061975 399 3 chr16: 32118280 + 32118678 399 4 chr2: 92264835 + 92265233 399 5 chr2: 91988419 + 91988817 399 6 chr2: 91660815 + 91661212 398 7 chr2: 90438986 + 90439383 398 8 chr7: 64574786 + 64575183 398 9 chr7: 61674980 + 61675378 399 10 chr9: 42734709 + 42735107 399 11 chr10: 42604376 − 42604773 398 12 chr16: 46469585 − 46469982 398 13 chr18: 15206512 − 15206910 399 14 chr18: 15162997 − 15163395 399 15 chr21: 10893308 − 10893706 399 16 chr2: 132804633 − 132805031 399 17 chr2: 132765347 − 132765745 399 18 chr2: 92240009 − 92240407 399 19 chr7: 65046103 − 65046500 398 20 chr7: 64983213 − 64983610 398 21 chr7: 61761667 − 61762065 399 22 chr7: 61058228 − 61058626 399 23 chr7: 57937338 − 57937736 399 24 chr9: 70411858 − 70412256 399 25 chr9: 70160930 − 70161328 399 26 chr9: 69795930 − 69796328 399

TABLE 13 Targets for Primer Assay No. 12 Hit No. Hit Location (hg19) Hit Size (bp) 1 chr10: 99459976 + 99460376 401 2 chr10: 98163398 + 98163798 401 3 chr10: 33183786 + 33184184 399 4 chr11: 123150791 + 123151191 401 5 chr11: 67689132 + 67689532 401 6 chr11: 37952518 + 37952918 401 7 chr12: 49509829 + 49510229 401 8 chr13: 43285312 + 43285712 401 9 chr21: 39945179 + 39945579 401 10 chr22: 17540425 + 17540825 401 11 chrUn_gl000223: 14920 + 15320 401 12 chrUn_gl000223: 9981 + 10381 401 13 chrUn_gl000231: 22879 + 23279 401 14 chr1: 236668200 + 236668600 401 15 chr1: 47599249 + 47599648 400 16 chr1: 40937804 + 40938204 401 17 chr2: 95857490 + 95857890 401 18 chr3: 75587273 + 75587673 401 19 chr4: 122318082 + 122318482 401 20 chr4: 9753064 + 9753464 401 21 chr4: 4076419 + 4076819 401 22 chr5: 161797510 + 161797907 398 23 chr6: 52748434 + 52748834 401 24 chr7: 104789485 + 104789885 401 25 chr8: 12423891 + 12424287 397 26 chr8: 8063597 + 8063995 399 27 chr9: 131612897 + 131613292 396 28 chr9: 129479179 + 129479579 401 29 chr9: 28880591 + 28880991 401 30 chrX: 153749772 + 153750170 399 31 chrX: 90425667 + 90426067 401 32 chr10: 96957178 − 96957577 400 33 chr11: 118879040 − 118879439 400 34 chr11: 71384933 − 71385333 401 35 chr11: 67597682 − 67598080 399 36 chr11: 23114276 − 23114676 401 37 chr11: 3468882 − 3469280 399 38 chr12: 133672280 − 133672680 401 39 chr12: 133667345 − 133667745 401 40 chr12: 8584230 − 8584630 401 41 chr13: 114947613 − 114948011 399 42 chr14: 106487687 − 106488087 401 43 chr15: 98172559 − 98172959 401 44 chr15: 90890020 − 90890420 401 45 chr1: 113359623 − 113360023 401 46 chr3: 142471114 − 142471514 401 47 chr3: 98081627 − 98082027 401 48 chr3: 15187255 − 15187655 401 49 chr4: 157307733 − 157308132 400 50 chr4: 43003876 − 43004276 401 51 chr5: 154023434 − 154023835 402 52 chr5: 152177074 − 152177473 400 53 chr6: 52812723 − 52813123 401 54 chr6: 43207456 − 43207860 405 55 chr7: 67568967 − 67569365 399 56 chr8: 12316718 − 12317116 399 57 chr8: 12074196 − 12074594 399 58 chr8: 7556947 − 7557347 401 59 chr8: 7098403 − 7098803 401 60 chr8: 6984728 − 6985128 401

The locations and sizes of additional hits with less stringent criteria (i.e., having perfect matches with 15 nucleotides at the 3′ end of primers) for some of the twelve primer pairs are provided in Table 14 below.

TABLE 14 Additional Targets for Primer Assays Assay Additional Hit Size No. Hit No. Hit Location (hg19) (bp) 2 1 chr1: 149030745 + 149030847 103 2 chr7: 53197939 − 53198041 103 3 1 chr16: 57432726 + 57432817 92 2 chr8: 39738896 + 39738987 92 3 chrX: 111355904 + 111355995 92 4 chr17: 50654589 − 50654680 92 5 chr17: 26042855 − 26042946 92 6 chr2: 148072985 − 148073076 92 7 chr4: 185659639 − 185659730 92 8 chr5: 99320377 − 99320468 92 5 1 chr21: 9475041 + 9475252 212 6 1 chr10: 33184168 + 33184386 219 2 chr11: 123151175 + 123151393 219 3 chr12: 8417305 − 8417523 219 4 chr15: 40936175 − 40936393 219 5 chr1: 147181566 − 147181784 219 8 1 chr7: 56901030 + 56901336 307 2 chr7: 51460780 + 51461085 306 9 1 chr7: 53197907 − 53198201 295 10 1 chr1: 243220852 + 243221231 380 2 chr1: 222650775 + 222651154 380 3 chr4: 120331573 + 120331952 380 4 chr7: 51460707 + 51461085 379 5 chr10: 38736084 − 38736463 380 6 chr4: 119550936 − 119551315 380 7 chr7: 128290584 − 128290963 380 12 1 chr2: 94434 + 94835 402 2 chr5: 24466249 + 24466649 401 3 chrY: 10013901 + 10014301 401 4 chr10: 102793517 − 102793917 401 5 chr11: 3416009 − 3416409 401 6 chr14: 20709942 − 20710342 401 7 chr16: 10923540 − 10923940 401 8 chr2: 702776 − 703176 401 9 chr3: 121330178 − 121330561 384 10 chr4: 128360853 − 128361253 401 11 chr4: 122367400 − 122367800 401 12 chr4: 3907275 − 3907674 400 Methods for Performing PCR Assay

Genomic DNA was extracted from human FFPE samples (normal human genomic DNA from Promega (Cat. No. G304X, 90% of the DNA is longer than 50 kb in size as measured by pulsed-field gel electrophoresis) was used as control). 1-2 ng of genomic DNA was used in each PCR reaction. DNA Quality Control (QC) PCR Array and Qiagen RT² Real-Timer SYBR Green/ROX PCR Mix were used to perform PCR. The 12 primer pair assays of the DNA QC PCR Array were predispensed in 384-well PCR plate as layout in FIG. 1.

Methods for Performing Next Generation Sequencing

Briefly, a multiplexed PCR assay that targeted 400 amplicons was developed for target enrichment. Primers were designed to avoid known SNPs and repetitive sequences. 20 ng of normal human genomic DNA or genomic DNA isolated from FFPE samples were evaluated for multiplexed PCR based target enrichment. PCR enriched samples were subjected to NGS library construction with Ion Xpress Plus Fragment Library Kit. After quantification with GeneRead Library Quantification Array, template (library) dilution factor was determined for each sample. Appropriately diluted prepared NGS libraries were sequenced on Ion Torrent PGM sequencer according to Life Technologies' user guide.

In a preliminary study, DNA from seven FFPE samples of varying quality and control Universal DNA were analyzed in duplicate by real-time PCR using 12 selected qPCR primer assays. The average Ct values of all three sets of 100 bp, 200 bp, 300 bp and 400 bp amplicons were determined for each DNA sample. The average Ct value of specific amplicon size for the control DNA was subtracted from average Ct value of that amplicon size for FFPE DNA.

The resulting ΔCt values were plotted against the outcome of sequencing results (FIG. 2). Samples with ΔCt values lower than 5, 10, 15 and 17 with 100 bp, 200 bp, 300 bp and 400 bp amplicons, respectively, showed good sequencing results (FIG. 2). This suggests that selected 12 qPCR primer assays can be used to pre-qualify DNA samples for successful sequencing results.

Five out of seven FFPE samples generated successful sequencing results. One sample (FFPE-1) generated smaller read length as compared to control universal DNA as well as other analyzed samples (FIG. 3), suggesting low quality of starting DNA with a potential risk of amplicon bias. The FFPE-7 sample showed insufficient amount of amplifiable library molecules upon qPCR based library quantification, after NGS library preparation, and did not process for sequencing.

Assuming that the higher the library dilution factor, the better the quality of DNA, samples with successful sequencing results were ranked for DNA quality as shown in Table 15 below. In addition, the average Ct value for the 12 assays of DNA QC Array was used to rank the DNA quality, assuming that the lower the averaged Ct value, the higher the quality rank. Interestingly, quality ranks based on the library dilution factors and based on the DNA QC Array analysis are the same. This suggests that the DNA QC Array analysis may be useful for recommending appropriate DNA input amount for NGS.

TABLE 15 Library dilution Quality rank based Quality rank based on Samples factor on dilution factor DNA QC Panel results FFPE-2 16.0 1 1 FFPE-3 4.3 5 5 FFPE-4 10.5 4 4 FFPE-5 12.3 3 3 FFPE-6 14.8 2 2

The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure. 

The invention claimed is:
 1. A method for assessing the quality of a test genomic DNA sample for a downstream DNA analysis, comprising: (a) performing one or more real-time PCR reactions that use genomic DNA in a test genomic DNA sample as templates in the presence of two or more primer pairs, wherein each of the two or more primer pairs is specific for amplifying identical or nearly identical genomic DNA fragments that are present at 10 or more locations in the genome of the organism from which the DNA sample is obtained, and wherein at least one of the two or more primer pairs is selected from the following primer pairs: (1) SEQ ID NOS:1 and 2, (2) SEQ ID NOS:3 and 4, (3) SEQ ID NOS:5 and 6, (4) SEQ ID NOS:7 and 8, (5) SEQ ID NOS:9 and 10, (6) SEQ ID NOS:11 and 12, (7) SEQ ID NOS:13 and 14, (8) SEQ ID NOS:15 and 16, (9) SEQ ID NOS:17 and 18, (10) SEQ ID NOS:19 and 20, (11) SEQ ID NOS:21 and 22, and (12) SEQ ID NOS:23 and
 24. (b) performing one or more real-time PCR reactions that use genomic DNA in a control genomic DNA sample as templates in the presence of the two or more primer pairs used in step (a), (c) determining the Ct values for the one or more real-time PCR reactions in step (a), (d) determining the Ct values for the one or more real-time PCR reactions in step (b), (e) assessing the quality of the test genomic DNA sample by determining the differences (ΔCt) between the Ct values determined in step (c) and the corresponding Ct values determined in step (d) for the one or more real-time PCR reactions, and (f) performing one or more additional DNA analyses of the test genomic DNA sample whose quality has been assessed in step (e).
 2. The method of claim 1, wherein the number of the primer pairs is 4-8.
 3. The method of claim 1, wherein the genomic DNA fragments amplified by the two or more primer pairs are present in more than 50%, 60%, 70%, or 80% of all autosomes of the organism from which the DNA sample is obtained.
 4. The method of claim 1, wherein the test genomic DNA sample is obtained from human cells or tissue.
 5. The method of claim 4, wherein the test genomic DNA sample is obtained from a clinical sample.
 6. The method of claim 1, wherein the test genomic DNA sample is obtained from a formalin fixed and paraffin-embedded (FFPE) sample.
 7. The method of claim 1, wherein the genomic DNA fragments amplified in step (a) are between about 100 to 400 bp in length.
 8. The method of claim 1, wherein the genomic DNA fragments amplified in step (a) are of at least 2 substantially different sizes.
 9. The method of claim 1, wherein multiple real-time PCR reactions are performed in each of steps (a) and (b), and assessing the quality of the test genomic DNA sample of step (e) is performed by determining the average difference between the Ct values determined in step (c) and the corresponding Ct values determined in step (d) for the multiple real-time PCR reactions.
 10. The method of claim 1, wherein the two or more primer pairs comprise the following primer pairs: (1) SEQ ID NOS:1 and 2, (2) SEQ ID NOS:3 and 4, (3) SEQ ID NOS:5 and 6, (4) SEQ ID NOS:7 and 8, (5) SEQ ID NOS:9 and 10, (6) SEQ ID NOS:11 and 12, (7) SEQ ID NOS:13 and 14, (8) SEQ ID NOS:15 and 16, (9) SEQ ID NOS:17 and 18, (10) SEQ ID NOS:19 and 20, (11) SEQ ID NOS:21 and 22, and (12) SEQ ID NOS:23 and
 24. 11. The method of claim 1, wherein in step (f), an additional real-time PCR and/or NGS analysis of the test genomic DNA sample is performed.
 12. The method of claim 1, wherein step (f) comprises: (i) measuring PCR amplifiable molecules in the test genomic DNA sample using the difference (ΔCt) determined in step (e); and (ii) determining the input amount of the test genomic DNA for an additional real-time PCR reaction based on the ΔCt.
 13. The method of claim 1, wherein the genomic DNA fragments amplified in steps (a) and (b) are not present on sex chromosomes.
 14. The method of claim 1, wherein no more than 50%, 40%, 30%, or 20% of the genomic DNA fragments amplified in steps (a) and (b) are located on a single chromosome.
 15. The method of claim 1, wherein the genomic DNA fragments amplified by a particular primer pair in steps (a) and (b) are present at 3 or more different autosomes.
 16. The method of claim 1, wherein no more than 50%, 40%, 30% or 20% of the genomic DNA fragments amplified by a particular primer pair are located on a single chromosome.
 17. The method of claim 1, wherein the number of the two or more primer pairs is 2-12.
 18. The method of claim 8, wherein assessing the quality of the test genomic DNA sample of step (e) further comprises determining amplification efficiency and amount or size distribution of amplifiable DNA fragments in the test genomic DNA sample.
 19. The method of claim 8, further comprising comparing the performance of PCR assays generating amplicons of different sizes and evaluating the distribution of amplifiable fragments in the test genomic DNA sample.
 20. The method of claim 1, wherein the organism from which the test genomic DNA sample is obtained is human, and wherein the identical or nearly identical genomic DNA fragments of step (a) are selected from the targets with chromosomal locations shown in Tables 2-14.
 21. The method of claim 12, wherein the input amount of the test genomic DNA sample in the additional real-time PCR is determined based on the ΔCt determined in step (e) for the additional real-time PCR.
 22. The method of claim 1, wherein each of the two or more primer pairs is included in an individual real-time PCR reaction or wherein the two or more primer pairs are included in a single real-time PCR reaction. 